GPx3 deficiency exacerbates maladaptive right ventricular remodeling in experimental pulmonary artery banding.

The stressed right ventricle (RV) is particularly susceptible to producing and accumulating reactive oxygen species, leading to extracellular matrix deposition and secretion of natriuretic peptides. The role of specific enzymes with antioxidative capacity, like glutathione peroxidase 3 (GPx3), in RV pathogenesis is currently unknown. Here, we use a murine model of pulmonary artery banding (PAB) to study the role of GPx3 in isolated RV pathology. Compared with wild-type (WT) mice undergoing PAB surgery, GPx3-deficient PAB mice presented with higher RV systolic pressure and higher LV eccentricity indices. PAB-induced changes in Fulton's Index, RV free wall thickness, and RV fractional area change were more pronounced in GPx3-deficient mice compared with WT controls. Adverse RV remodeling was enhanced in GPx3-deficient PAB animals, evidenced by increased RV expression levels of connective tissue growth factor (CTGF), transforming growth factor-ß (TGF-ß), and atrial natriuretic peptide (ANP). In summary, GPx3 deficiency exacerbates maladaptive RV remodeling and causes signs of RV dysfunction.

L-2-Hydroxyglutarate Protects Against Cardiac Injury via Metabolic Remodeling.

BACKGROUND: L-2-hydroxyglutarate (L2HG) couples mitochondrial and cytoplasmic energy metabolism to support cellular redox homeostasis. Under oxygen-limiting conditions, mammalian cells generate L2HG to counteract the adverse effects of reductive stress induced by hypoxia. Very little is known, however, about whether and how L2HG provides tissue protection from redox stress during low-flow ischemia (LFI) and ischemia-reperfusion injury. We examined the cardioprotective effects of L2HG accumulation against LFI and ischemia-reperfusion injury and its underlying mechanism using genetic mouse models. METHODS AND RESULTS: L2HG accumulation was induced by homozygous (L2HGDH [L-2-hydroxyglutarate dehydrogenase]-/-) or heterozygous (L2HGDH+/-) deletion of the L2HGDH gene in mice. Hearts isolated from these mice and their wild-type littermates (L2HGDH+/+) were subjected to baseline perfusion and 90-minute LFI or 30-minute no-flow ischemia followed by 60- or 120-minute reperfusion. Using [13C]- and [31P]-NMR (nuclear magnetic resonance) spectroscopy, high-performance liquid chromatography, reverse transcription quantitative reverse transcription polymerase chain reaction, ELISA, triphenyltetrazolium staining, colorimetric/fluorometric spectroscopy, and echocardiography, we found that L2HGDH deletion induces L2HG accumulation at baseline and under stress conditions with significant functional consequences. In response to LFI or ischemia-reperfusion, L2HG accumulation shifts glucose flux from glycolysis towards the pentose phosphate pathway. These key metabolic changes were accompanied by enhanced cellular reducing potential, increased elimination of reactive oxygen species, attenuated oxidative injury and myocardial infarction, preserved cellular energy state, and improved cardiac function in both L2HGDH-/- and L2HGDH+/- hearts compared with L2HGDH+/+ hearts under ischemic stress conditions. CONCLUSION: L2HGDH deletion-induced L2HG accumulation protects against myocardial injury during LFI and ischemia-reperfusion through a metabolic shift of glucose flux from glycolysis towards the pentose phosphate pathway. L2HG offers a novel mechanism for eliminating reactive oxygen species from myocardial tissue, mitigating redox stress, reducing myocardial infarct size, and preserving high-energy phosphates and cardiac function. Targeting L2HG levels through L2HGDH activity may serve as a new therapeutic strategy for cardiovascular diseases related to oxidative injury.

Interferon-γ Impairs Human Coronary Artery Endothelial Glucose Metabolism by Tryptophan Catabolism and Activates Fatty Acid Oxidation.

BACKGROUND: Endothelial cells depend on glycolysis for much of their energy production. Impaired endothelial glycolysis has been associated with various vascular pathobiologies, including impaired angiogenesis and atherogenesis. IFN-γ (interferon-γ)-producing CD4+ and CD8+ T lymphocytes have been identified as the predominant pathological cell subsets in human atherosclerotic plaques. Although the immunologic consequences of these cells have been extensively evaluated, their IFN-γ-mediated metabolic effects on endothelial cells remain unknown. The purpose of this study was to determine the metabolic consequences of the T-lymphocyte cytokine, IFN-γ, on human coronary artery endothelial cells. METHODS: The metabolic effects of IFN-γ on primary human coronary artery endothelial cells were assessed by unbiased transcriptomic and metabolomic analyses combined with real-time extracellular flux analyses and molecular mechanistic studies. Cellular phenotypic correlations were made by measuring altered endothelial intracellular cGMP content, wound-healing capacity, and adhesion molecule expression. RESULTS: IFN-γ exposure inhibited basal glycolysis of quiescent primary human coronary artery endothelial cells by 20% through the global transcriptional suppression of glycolytic enzymes resulting from decreased basal HIF1α (hypoxia-inducible factor 1α) nuclear availability in normoxia. The decrease in HIF1α activity was a consequence of IFN-γ-induced tryptophan catabolism resulting in ARNT (aryl hydrocarbon receptor nuclear translocator)/HIF1ß sequestration by the kynurenine-activated AHR (aryl hydrocarbon receptor). In addition, IFN-γ resulted in a 23% depletion of intracellular nicotinamide adenine dinucleotide in human coronary artery endothelial cells. This altered glucose metabolism was met with concomitant activation of fatty acid oxidation, which augmented its contribution to intracellular ATP balance by >20%. These metabolic derangements were associated with adverse endothelial phenotypic changes, including decreased basal intracellular cGMP, impaired endothelial migration, and a switch to a proinflammatory state. CONCLUSIONS: IFN-γ impairs endothelial glucose metabolism by altered tryptophan catabolism destabilizing HIF1, depletes nicotinamide adenine dinucleotide, and results in a metabolic shift toward increased fatty acid oxidation. This work suggests a novel mechanistic basis for pathological T lymphocyte-endothelial interactions in atherosclerosis mediated by IFN-γ, linking endothelial glucose, tryptophan, and fatty acid metabolism with the nicotinamide adenine dinucleotide balance and ATP generation and their adverse endothelial functional consequences.

Nucleophosmin Phosphorylation as a Diagnostic and Therapeutic Target for Ischemic AKI.

Background Ischemic AKI lacks a urinary marker for early diagnosis and an effective therapy. Differential nucleophosmin (NPM) phosphorylation is a potential early marker of ischemic renal cell injury and a therapeutic target.Methods Differential NPM phosphorylation was assessed by mass spectrometry in NPM harvested from murine and human primary renal epithelial cells, fresh kidney tissue, and urine before and after ischemic injury. The biologic behavior and toxicity of NPM was assessed using phospho-NPM mutant proteins that either mimic stress-induced or normal NPM phosphorylation. Peptides designed to interfere with NPM function were used to explore NPM as a therapeutic target.Results Within hours of stress, virtually identical phosphorylation changes were detected at distinct serine/threonine sites in NPM harvested from primary renal cells, tissue, and urine. A phosphomimic NPM protein that replicated phosphorylation under stress localized to the cytosol, formed monomers that interacted with Bax, a cell death protein, coaccumulated with Bax in isolated mitochondria, and significantly increased cell death after stress; wild-type NPM or a phosphomimic NPM with a normal phosphorylation configuration did not. Three renal targeted peptides designed to interfere with NPM at distinct functional sites significantly protected against cell death, and a single dose of one peptide administered several hours after ischemia that would be lethal in untreated mice significantly reduced AKI severity and improved survival.Conclusions These findings establish phosphorylated NPM as a potential early marker of ischemic AKI that links early diagnosis with effective therapeutic interventions.

Proteinuria causes dysfunctional autophagy in the proximal tubule.

Proteinuria is a major risk factor for chronic kidney disease progression. Furthermore, exposure of proximal tubular epithelial cells to excess albumin promotes tubular atrophy and fibrosis, key predictors of progressive organ dysfunction. However, the link between proteinuria and tubular damage is unclear. We propose that pathological albumin exposure impairs proximal tubular autophagy, an essential process for recycling damaged organelles and toxic intracellular macromolecules. In both mouse primary proximal tubule and immortalized human kidney cells, albumin exposure decreased the number of autophagosomes, visualized by the autophagosome-specific fluorescent markers monodansylcadaverine and GFP-LC3, respectively. Similarly, renal cortical tissue harvested from proteinuric mice contained reduced numbers of autophagosomes on electron micrographs, and immunoblots showed reduced steady-state LC3-II content. Albumin exposure decreased autophagic flux in vitro in a concentration-dependent manner as assessed by LC3-II accumulation rate in the presence of bafilomycin, an H+-ATPase inhibitor that prevents lysosomal LC3-II degradation. In addition, albumin treatment significantly increased the half-life of radiolabeled long-lived proteins, indicating that the primary mechanism of degradation, autophagy, is dysfunctional. In vitro, mammalian target of rapamycin (mTOR) activation, a potent autophagy inhibitor, suppressed autophagy as a result of intracellular amino acid accumulation from lysosomal albumin degradation. mTOR activation was demonstrated by the increased phosphorylation of its downstream target, S6K, with free amino acid or albumin exposure. We propose that excess albumin uptake and degradation inhibit proximal tubule autophagy via an mTOR-mediated mechanism and contribute to progressive tubular injury.